Abstract

Refilins (RefilinA and RefilinB) are members of a novel family of Filamin binding proteins that function as molecular switches to conformationally alter the Actin filament network into bundles. We show here that Refilins are extremely labile proteins. An N-terminal PEST/DSG(X)2-4S motif mediates ubiquitin-independent rapid degradation. A second degradation signal is localized within the C-terminus. Only RefilinB is protected from rapid degradation by an auto-inhibitory domain that masks the PEST/DSG(X)2-4S motif. Dual regulation of RefilinA and RefilinB stability was confirmed in rat brain NG2 precursor cells (polydendrocyte). Using loss- and gain-of-function approaches we show that in these cells, and in U373MG cells, Refilins contribute to the dynamics of lamellipodium protrusion by catalysing Actin bundle formation within the lamella Actin network. These studies extend the Actin bundling function of the Refilin-Filamin complex to dynamic regulation of cell membrane remodelling.

KEYWORDS:

Refilins are short-lived proteins. (A) Sequence alignment of the N-terminus of rat RefilinA (residues 1-99) and RefilinB (residues 1-112) proteins show conserved N-terminal sequence harbouring a PEST/DSG(X)2-4S motif (PEST). The specific adjacent sequence only found in RefilinB is squared. (B,C) Cycloheximide chase analysis of full­-length and truncated 50-204 Myc-tagged RefilinA proteins expressed in sub-confluent U373MG cells. Transfected cells were incubated with cycloheximide (100 µg/ml). Cell extracts were resolved on 12% SDS-PAGE and analysed by western blot using chicken anti-RefilinA or mouse anti-Vimentin as loading control. (C) Quantitative analysis of the western blot shown in panel B of two independent cycloxeximide chase experiments. The mean±standard error (s.e.m.) of two different experiments are shown, statistically different from control condition (Student's t-test, P<0.009). (D) Sub-confluent U373MG cells transfected with Myc-tagged RefilinB (upper panel) and truncated Δ40-65 RefilinB (lower panel). After 20 h cells were incubated with cycloheximide (100 µg/ml) for the indicated times. Cell extracts were analysed by western blot using mouse anti-Myc monoclonal antibody. Asterisks point to a non-specific band specifically recognized by the mouse anti-Myc monoclonal antibody (see also E,F). (E) Comparison of the steady-state level of Myc-tagged RefilinA (lane 2), Myc-tagged truncated Δ10-35-RefilinA (lane 3), Myc-tagged RefilinB (lane 4), and Myc-tagged truncated Δ40-65-RefilinB (lane 5) expressed in U373MG cells. Sub-confluent U373MG cells in 100 mm plates were transfected with 2 µg of recombinant pcDNA-3 plasmids and incubated 20 h prior analyses. Lane 1 is a non-transfected control cell lysate. Cell extracts were analysed by western blot using anti-Myc. Asterisks point to a non-specific band specifically recognized by the mouse anti-Myc monoclonal antibody. (F) Sub-confluent U373MG cells were transfected with Myc-tagged RefilinA, truncated 50-204 RefilinA (Δ1-50) and RefilinB constructs as indicated. After 20 h, transfected cells were incubated with protease inhibitor MG132 (5 µM) for the indicated times. Cell extracts were analysed by western blot using rat anti-Myc and anti-α-tubulin. The non-specific band recognized by the mouse anti-Myc monoclonal antibody (* in D,E) is not recognized by the rat anti-Myc antibody. (G) Sub-confluent U373MG cells transfected with RefilinA-GFP were incubated with cycloheximide (100 µg/ml) in the absence or in the presence of MG132 or Calpeptin for the indicated times. Total cell extracts were analysed by western blot using mouse anti-GFP antibody.

RefilinB regulates the dynamic of lamellipodia in NG2 cells. (A) OPC were infected with recombinant adenovirus expressing GFP-control (lane 1), ShRefilinB (lane 2) or RefilinB-GFP (lane 3) and grown for 24 h in differentiation culture medium. Total cell extracts were analysed by western blot using anti-RefilinB antibodies. Arrows point to specific RefilinB immunoreactivities, asterisks indicate non-specific bands. (B) Video recordings of OPC infected with recombinant adenovirus expressing GFP-control (GFP), ShRefilinB or RefilinB-GFP were used to quantify the percentage of processes that form lamellipodia during the time of recording (10 min). The results are the average of two infection experiments and the analyses of 100 processes per experiment. Error bars represent the variability of data between two independent experiments

RefilinA enhances spreading of U373MG cells. U373MG cells were infected with recombinant adenovirus expressing GFP-control (A) or GFP-RefilinA (B). After 24 h, cell spreading was quantified by allowing the cells to spread on fibronectin-coated coverslips for 20 min. Cells attached to coverslips were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 3 min. Cells were blocked with 3% BSA in PBS containing 0.1% Tween 20, and incubated with Alexa Fluor 486 Phalloidin for 1 h. Cells were mounted and visualized under a confocal microscope at low magnification (A,B). Scale bar: 50 µm. On the right side the percentage of cells adopting spread morphology is reported. Non-spread cells were defined as round cells, whereas spread cells were defined as those that lacked a rounded shape and had extended membrane protrusions. In each experiment, more than 150 cells were counted. Two independent experiments were performed.